Published on Web 05/16/2003
Novel Selective Inhibitors of the Interaction of Individual Nuclear Hormone
Receptors with a Mutually Shared Steroid Receptor Coactivator 2
Timothy R. Geistlinger and R. Kiplin Guy*
Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, UniVersity of California at
San Francisco, Genentech Hall, Mission Bay, 600 16th Street 2280, San Francisco, California 94143-2280
Received February 24, 2003; E-mail: rguy@cgl.ucsf.edu
The nuclear hormone receptor transcription factors (NRs) respond
directly to small-molecule ligands and control diverse functions in
vivo, including development and dynamic homeostasis.1,2 Both
metabolic diseases and cancer have been directly correlated with
the misregulation of signaling by the NRs, and they are the target
for multiple drugs and drug development programs.3 Ligand-
dependent NR signaling requires direct interaction between NRs
and the steroid receptor coactivators (SRCs), effected by a conserved
SRC motif (NR box, L1XXL2L3).1 We have previously described
potent but nonselective inhibitors of the interaction of NRs and
SRCs. Using computational and parallel synthetic techniques, we
have produced a library of novel proteomimetics of the second NR
box of SRC2 (SRC2-2) that exploit structural differences between
the thyroid hormone receptor (TR) and two isoforms of the estrogen
receptor (ER) to selectively inhibit the interaction of SRC2-2 with
human estrogen receptor R (hERR), human estrogen receptor â
smoothly to yield milligram quantities of each compound after RP-
HPLC purification. Many of the CR-branched compounds proved
to be more difficult to synthesize and purify, particularly during
macrolactam formation. The final proteomimetic library contained
87 of 111 targeted compounds composed of 25, 30, and 32
individual substitutions, respectively, at positions L1, L2, and L3
(Figure 1C). The purity and identity of all compounds were
confirmed by LCMS and HR-MALDI-TOF MS, respectively.
The ability of the proteomimetics to compete with SRC2-2
peptide for binding to the NR was assessed using fluorescence
polarization (FP) equilibrium competition assays.7 These studies
revealed that the library members exhibited a large range of
inhibitory ability for blocking the binding of the SRC2-2 peptide
to the NR (Figure 1C). As hypothesized from the DOCKing studies,
the majority of phenylglycine and phenylalanine analogues allowed
for effective competition, with 71 of 87 compounds giving IC50’s
that were equivalent to or better than those of the SRC2-2 peptide
with one or more NR. The high level of success in picking
compounds with competitive ability validates the use of the
CombiDOCK methodology for the design of inhibitors of protein-
protein interactions in addition to its conventional use in design of
enzyme inhibitors.
The testing of the inhibitors identified the first selective proteo-
mimetics (Figure 1D) that take advantage of differences between
the leucine binding pockets of E2‚hERR, E2‚hERâ, and T3‚hTRâ.
A total of 12 compounds were at least 10-fold selective for binding
to hERR in preference to hTRâ or hERâ (Figure 1D). Of these, a
number were more than 20-fold selective, and one, 1{34,37,37},
was more than 600-fold selective for hERR. Surprisingly, only one
compound, 1{3,37,37}, was selective for hTRâ and one, 1{37,14,37},
for hERâ. Strikingly, two natural amino acids, tryptophan and
isoleucine, provide high levels of selectivity for hERR. Previous
studies using genetic selection from random peptide libraries
failed to reveal this trend, instead selecting exclusively for leucines
within the L1XXL2L3 motif while evolving differences in flanking
sequence.8
(hERâ), and human thyroid hormone receptor
â (hTRâ)
individually.
Structurally, hERR and hTRâ interact with the SRC2-2 peptide
through similar surfaces, with a shallow hydrophobic groove on
the surface binding to an amphipathic R-helical motif on the
SRC2-2, burying the three leucines on the hydrophobic face of
the NR box helix.4,5 To explore the possibility of discovering
coactivator binding inhibitors selective to particular NR‚ligand pairs,
we designed a library of R-helical proteomimetics, constrained
by a macrolactam at positions E691 and K695, that mimic SRC2-2
{c(E691-K695)Ac-685EKHKIL1ERL2L3KDS697-COOH} (1{37,37,37},
Figure 1A) with non-natural amino acids (Figure 1B) replacing the
leucines at positions L1, L2, and L3. The selection of particular non-
natural side chains for this library was governed by computational
DOCKing (CombiDOCK)6 methods using hERR‚E2‚SRC2-2 and
hTRâ‚SRC2-2·T3 X-ray crystal structures. Hydrophobic, non-natural
amino acids in the Available Chemical Directory were substituted
in silico for each leucine by replacing the CR and Câ atom positions
of the crystallographic SRC2-2 peptide with those of the introduced
side chains. The resulting molecules were screened in silico against
the coactivator binding surfaces of the receptors and reduced to a
set of 45 diversity elements (Figure 1B) at each position for library
synthesis. Computationally, the NRs selected for similar residues
at each position, except that hERR tolerated larger and CR-branched
residues (Figure 1B, 24, 25, 27-34). Studies were carried out in
which each leucine was replaced individually without changing any
other amino acids and in which all three leucines were simulta-
neously varied. As the CombiDOCK studies did not reveal
significant cooperativity between the diversity positions, the targeted
library was limited to a single substitution in each member.
The designed library was synthesized in parallel on solid support
utilizing Fmoc chemistry as previously described for constrained
SRC2-2 mimetic 1{37,37,37}.7 In general, the synthesis proceeded
The SRC binding pockets of NR have evolved to bind to a simple
hydrophobic L1XXL2L3 consensus motif while relying upon dif-
ferences in SRC structure flanking the NR box to convey selectivity
in vivo. This study reveals that the SRC binding pockets contain
significant differences in shape and electrostatics that allow
competitive inhibitors that mimic the NR box to act selectively on
one NR. The fact that discrimination between NRs can be achieved
solely by manipulation of the side chains inside the small L1XXL2L3
motif implies that a suitable small molecule could achieve the same
result. Thus, targeting this site may prove to be a general method
for producing selective modulators of NR function.
9
6852
J. AM. CHEM. SOC. 2003, 125, 6852-6853
10.1021/ja0348391 CCC: $25.00 © 2003 American Chemical Society